Tuberculosis (TB) remains a significant threat to international public health, as over 2 million individuals die of the disease each year. Mycobacterium tuberculosis (Mtb), the organism that causes TB, is a respiratory pathogen spread via inhalation of infectious airborne particles. Better understanding of local immunity to Mtb within the human lung is critical to development of more effective TB vaccination programs, and, more generally, to understanding the mechanisms by which cell-mediated immunity functions within the human lung. Our research team represents a unique collaboration of investigators with experience in bronchoscopy-based studies of human immunity to Mtb (Richard Silver, Case Western Reserve University) assessment of immune responses to experimental TB vaccination (Daniel Hoft, Saint Louis University) and immunological bioinformatics analysis (Vladimir Brusic, Dana Farber Cancer Institute). The overall goals of this proposal are to define the mechanisms that mediate protective local immunity to Mtb within the human lung, and to clarify the basis for the suboptimal efficacy of current TB vaccination with the attenuated Mycobacterium bovis strain the Bacillus of Calmette and Guerin (BCG). Understanding immunity to Mtb within the lung is of particular importance because although current intradermal (ID) BCG vaccination protects against disseminated forms of TB, it does not provide adequate protection against the most contagious form of the disease, pulmonary TB. We propose to apply genome-wide microarray analysis to evaluate the Mtb-induced transcriptome of cells obtained from the lung via bronchoalveolar lavage (BAL). Our preliminary studies indicate that global Mtb-induced gene expression of BAL cells from healthy subjects with latent tuberculosis infection (LTBI) following respiratory infection with the organism is markedly different from that of Mtb-naive subjects. These differences suggest that resident Mtb-specific effector memory T cells (TEM) present in baseline BAL have a profound impact on expression of the pulmonary transcriptome. This memory response includes promotion of rapid chemokine production as well as induction of genes involved in multiple processes that may potentially impact the viability of intracellular Mtb. We will also compare the Mtb-induced transcriptomes of baseline BAL cells from individuals with LTBI to those obtained from a unique cohort of subjects vaccinated with BCG either by the standard ID route, or by oral (PO) administration. In addition, we will assess the impact of this early chemokine production on recruitment of additional immune cells to the lung using a novel protocol of segmental bronchoscopic challenge with the skin-test reagent purified protein derivative of Mtb (PPD). PPD challenge serves as a model of local immune events following respiratory re-exposure to Mtb, and will allow us to compare the Mtb-induced transcriptome of these recruited cells to those of baseline BAL. Correlation of these gene expression findings with measures of inhibition of virulent Mtb will allow us determine the mechanisms by which cell recruitment enhances containment of Mtb within the lungs of both LTBI subjects and BCG vaccine recipients. These studies will therefore address the following Specific Aims: 1) To determine the mechanisms by which specific cytokines and T-cell populations contribute to the localized immunophenotype of pulmonary recall responses to Mycobacterium tuberculosis. 2) To evaluate potential mechanisms by which BCG vaccination may provide suboptimal development of M. tuberculosis-specific immunity within the human lung; and 3) To evaluate the processes by which cells recruited to the lung in response to re-exposure to M. tuberculosis antigens enhance containment of the organism in both Mtb-infected and BCG-vaccinated individuals.